Mitochondrial division is a fundamental process of vital importance for cell growth and survival. The mechanism of mitochondrial division is, however, poorly understood. In the current funding period, we discovered that a dynamin-related protein, which we call Drp1, is required for division of the mitochondrial outer membrane. We have new evidence indicating that endophilin, a protein known to induce membrane curvature at the necks of budding endocytic vesicles, also has a counterpart in mitochondrial division, named endophilin B in mammals and ERP-1 in C. elegans. The discovery that two very different endocytic proteins have counterparts in the mitochondrial division apparatus indicates that the machineries for these two processes arose from a common evolutionary ancestor. Given the complexity of the endocytic machinery, we expect to find additional proteins in the mitochondrial division complex as well. In Aim 1 we propose to identify proteins that contribute to mitochondrial outer membrane division in C. elegans. We will continue to investigate C. elegans ERP-1 and look for other factors that might contribute to mitochondrial division. New mitochondrial division proteins will be identified based on analogous functions of endocytic proteins, homologies to yeast mitochondrial division proteins and open-ended screens (bioinformatics, RNAi and classic genetic screens). In aim 2 we propose to analyze the functions of mammalian Erp1 and other mitochondrial division proteins in mammalian cells using well-established cell biological techniques. These experiments are likely to provide new insights, especially with the approaches that cannot be applied to worms. In Aim 3 we propose to characterize possible connections between mitochondrial division and apoptosis. These experiments are based on the recent discovery that mutant Drpl inhibits apoptosis and the discovery that mammalian endophilin B binds to Bax. The wealth of apoptosis mutants in C. elegans gives us a unique advantage to explore the relationship between mitochondrial division and apoptosis. In complementary experiments, we will explore the physical interactions between apoptosis and mitochondrial division machineries using mammalian biochemistry. Together, the proposed experiments will give a more complete understanding of the protein machineries that regulate and execute mitochondrial division.